Improvements in the Synthesis of Phycocyanins

ABSTRACT

The invention discloses microorganism cell culture conditions that result in increased cellular and media concentrations of a biological pigment. The invention has applications in use as a natural food colouring, as antioxidants in the food supplement industries, in the nutraceutical, pharmaceutical, and cosmeceutical industries, and a non-toxic ink. The method results in pigment that is relatively easy to separate from the microorganism culture.

RELATIONSHIP TO OTHER APPLICATIONS

This application claims priority to and benefits of the following: U.S.Provisional Patent Application No. 61/931,723 filed 27 Jan. 2014,entitled “Improvements in the Synthesis of Phycocyanin” and U.S.Provisional Patent Application No. 62/134,479 filed 17 Jun. 2014,entitled “Improvements in the Synthesis of Phycocyanins”, which are bothherein incorporated by reference in their entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to using microbial cell culture conditionsthat result in increased levels and concentrations of pigments.

BACKGROUND

Phycocyanin (PC) is a blue pigmented billiprotein, a chromophoreproduced in prokaryotic cyanobacteria as well as certain eukaryotes suchas the rhodophytes, cryptomonads and glaucocystophytes. PC isincreasingly being exploited as a natural food colouring, replacing thesynthetic dye Brilliant Blue FCF that has been associated with healthproblems; PC is particularly suited to this use because of its highsolubility in water and stability over a large pH range [1]. Inaddition, PC is used in the nutraceutical, pharmaceutical andcosmeceutical industries at higher purities for its anti-oxidant andanti-inflammatory properties, together with other associated healthbenefits [2-4]. PC in its more crude form is also used as an additive toanimal feeds to enhance the colour of ornamental fish and birds. At itshighest quality and purity PC is used in laboratory assay kits for itsfluorescent properties. There is also early but ongoing research intothe therapeutic properties of PC for medical use [5]. The PC market isin its infancy.

In cyanobacteria, PC is present in the thylakoid membrane complexed withthe other biliproteins including phycoerythrin (PE) and allophycocyanin(AP or APC) which together function as a light-harvesting apparatusknown as the phycobillisome [6]. The phycobillisome absorbs specificwavelengths of light that cannot be utilized by chlorophyll, therebyincreasing the efficiency of photosynthesis [7]. PC absorbs maximally at610-620 nm with PE (540-570 nm) and APC (650-655 nm) [6].

Cyanobacteria are widely used in aquaculture for PC production with theeukaryotes showing potential for future exploitation. Among thecyanobacteria the genus Arthrospira (formerly known as Spirulina andstill commercially known as ‘Spirulina’) is the most commonly culturedgenus; however, PC has been extracted from other genera such asAphanizomenon and Anabaena. The main species in culture are Arthrospiraplatensis and A. maxima. These are both filamentous cyanobacteria withspiral-shaped filaments or trichomes.

In addition to its high PC content, spirulina also contains high amountsof other nutraceuticals such as vitamins and PUFAs and is high in singlecell protein; as a result, PC is becoming of increasing commercialinterest in the West [1]. In the East and Africa however, Spirulina hasbeen used as a food for many centuries [9]. Spirulina biomass is asalable product alone, however pure phycocyanin, depending on purity hasa considerably higher market price.

As water molecules absorb in the far red region of light, limitations inthis wavelength for photosynthesis occur in the natural algaeenvironment [6]. Light scattering of shorter wavelengths also occurs bysuspended material resulting in the provision bias of blue-greenwavelength light to algae in nature. Therefore environmental factorsdetermine light availability and algae can adapt to utilize quality andquantity of light available.

Some cyanobacteria containing PE and PC exhibit a phenomenon calledcomplementary chromatic adaptation where PC:PE ratio is altered inresponse to different light regimes by modulating synthesis [10, 11].Recent research has shown that A. platensis can be manipulated in thepresence of certain wavelengths of light to increase production of PC.

By using light filters Walter et al. (2011) [12] demonstrated increasedPC purity under red light (600-700 nm). Earlier research by Wang et al.also found A. platensis biomass productivity was higher culturing underred light [13]. The calculations by Wang et al. demonstrated that theuse of red light would be economically beneficial to photoautotrophicproduction, as energy to biomass conversion is more efficient. Farges,2009 [19] also modeled growth of A. platensis under polychromatic andmonochromatic light sources, demonstrating mathematical increases inculturing efficiency under red 620 nm LEDs through decreased electricalenergy power consumption with maintained and comparable growth rates;however monochromatic red LED light (620 nm) was shown to decrease thePC concentration of A. platensis by 2-fold, compared withwhite/polychromatic and red+blue polychromatic LEDs. These studies havedemonstrate that culturing under different wavelengths of light caneffect the PC concentration and purity in A. platensis cultures, howevertests have not been conducted using wavelengths above that of normal redLEDs.

There is therefore a need in the art for improved and less costlymethods for synthesis of phycocyanins.

SUMMARY OF THE INVENTION

The invention herein disclosed provides for devices and methods that maybe used for the improved synthesis of phycocyanins. The method resultsin a greater than 10-fold increase in phycocyanin levels, a clearimprovement over the prior art. The method also results in animprovement for harvesting phycocyanins.

The devices herein disclosed may be used in many applications,including, but not limited to, use as a natural food colouring, as anantioxidant in the food supplement industries, in the nutraceutical,pharmaceutical, and cosmeceutical industries, and as a non-toxic ink

The invention provides improved methods for the synthesis and commercialproduction of phycocyanins and other natural biochemical compositions,including but not limited to, hyaluronans, glucosamines, othersaccharides and/or polysaccharides, other phycobiliproteins, such as butnot limited to, allophycocyanin, phycoerythrin, bilin, phycobilin,proteoglycans, glycosaminoglycans, and the like.

In one embodiment, the method includes providing a microorganism capableof synthesizing phycocyanins, providing a suitable culture and growthmedium, illuminating the microorganism in culture with red and/ornear-infrared light, and in the alternative, illuminating themicroorganism in culture with red and/or near-infrared monochromaticlight. In an alternative embodiment, the method also providesilluminating the microorganism in culture with white light.

In another embodiment, the method includes providing an organism capableof photosynthesizing carbon-based compositions using energy from anatural or an artificial energy source. The organism may be aphotosynthetic bacterium, photosynthetic archaean, a photosyntheticprotist, a photosynthetic alga, a photosynthetic moss, or aphotosynthetic plant. The organism may be a naturally occurring speciesor it may be a synthetic organism created using recombinant DNAtechnology. The organism may be a domesticated plant species and mayalso comprise DNA from another organism.

In one embodiment the near-infrared light comprises electromagneticradiation having a wavelength between about 630 nm and about 720 nm. Inanother embodiment the near-infrared monochromatic light compriseselectromagnetic radiation having a wavelength of about 680 nm. In analternative embodiment the near-infrared monochromatic light compriseselectromagnetic radiation having a wavelength of about 678 nm. Inanother alternative embodiment the near-infrared monochromatic lightcomprises electromagnetic radiation having a wavelength of about 682 nm.In one embodiment the white light comprise electromagnetic radiationhaving wavelengths between about 350 nm and about 760 nm. In anotheralternative embodiment the near-infrared monochromatic light compriseselectromagnetic radiation having a wavelength of about 650 nm. In yetanother alternative embodiment the near-infrared monochromatic lightcomprises electromagnetic radiation having a wavelength of about 720 nm.In yet another alternative embodiment the monochromatic light compriseselectromagnetic radiation having a wavelength of between about 450 and590 nm.

In another embodiment the red light consists of electromagneticradiation having wavelengths between 640 nm and 720 nm. In anotherembodiment the red light consists of electromagnetic radiation havingwavelengths between 640 nm and 1000 nm. In another embodiment the redlight consists of electromagnetic radiation having a maximum wavelengthemission of 680 nm. In an alternative embodiment the red light consistsof electromagnetic radiation having a wavelength of 678 nm. In anotheralternative embodiment the red light consists of electromagneticradiation having a wavelength of 682 nm. In one embodiment the whitelight consists of electromagnetic radiation having wavelengths between350 nm and 760 nm.

In another preferred embodiment, the synthesized phycocyanin leachesfrom the microorganism.

In another embodiment, the microorganism capable of synthesizingphycocyanins is cultured in a pond system or open raceway system. In apreferred embodiment, >640 nm LED rods are placed in the pond system oropen raceway system and which results in increased synthesis ofphycocyanins in the microorganism.

In another embodiment the invention contemplates a system for producingphycocyanins, the system comprising a vessel and a lamp, wherein thelamp generates electromagnetic energy having a wavelength of at least640 nm or greater, and wherein the vessel further comprises amicroorganism capable of synthesizing phycocyanin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic of the energy conversion process in photosynthesis.P680 and P700 represent the reaction centre Ch1a of Photosystem II andPhotosystem I respectively. Phycocyanin (PC) (610-620 nm) is present ina complex with Phycoerythrin (PE) (540-570 nm) and Allophycocyanin (APC)for the phycobilisome light harvesting apparatus which absorb specificwavelengths of light for use in photosynthesis.

FIG. 2. The Infors Stirred Tank Photobioreactor system with aninterchangeable LED jacket.

FIG. 3. Typical emission spectra comparing typical white LED, typicalred LED (typically around 620-640 nm) and 680 nm LED light. Right showsemission spectra of 680 nm LED emitting narrow intense light with anoptimum emission wavelength of 680 nm.

FIG. 4. Growth curves for separate batch runs of A. platensis culture(error bars represent standard error of the mean) with table showingaverage growth rate of A. platensis under 680 nm LEDs compared totypical white LEDs, with no significant difference in growth under thetwo light conditions.

FIG. 5. Absorbance spectra of Phycocyanin extracts from A. platensis,normalized at 678 nm, cultured under 680 nm LEDs compared to typicalwhile LEDs. Light dashed lines represent error (s.e.m., standard errorof the mean). A larger absorption peak representing Phycocyanin can beseen at around 620 nm in the extract from A. platensis cultured under680 nm LEDs.

FIG. 6. Average Phycocyanin yield (mg/g) from A. platensis culturedunder 680 nm LEDs compared to typical white LEDs. Error bars representstandard error of the mean. A large significant increase in Phycocyaninlevels can be seen in A. platensis through culturing under 680 nm LEDs.

FIG. 7. Mass spectra for A. platensis cultures cultured under 680 nmLEDs compared to typical white LEDs show differences in abundantproteins under the two light conditions.

FIG. 8. Left shows A. platensis culture (top) and extract (bottom) fromculturing under typical white LED. Right shows A. platensis culture(top) and extract (bottom) from culturing under 680 nm LED.

FIG. 9. Average Phycocyanin yield (mg/g) for separate batches of A.platensis, inoculated from the previous batch, cultured under 680 nmLEDs shows an increasing yield of Phycocyanin with each subsequent run,likely indicating A. platensis is undergoing continual adaptation toenable utilization of 680 nm light more efficiently in photosynthesis.

FIG. 10. Growth curves for A. platensis batch cultures under 680 nmlight. Dashed line shows A. platensis culture undergoing acclimatizationfor utilization of 680 nm light. A lag phase where acclimatization isoccuring, is present up to day 14.

FIG. 11. Top left: Flocculation of higher Phycocyanin-yielding A.platensis cultured under 680 nm LEDs. Microscope images show presence ofcrystals on A. platensis trichomes in flocculated cultures (bottom),absent in non-flocculating culture, indicating increase in extracellularpolysaccharide, possibly as a stress response

GENERAL DISCLOSURES

The embodiments disclosed in this document are illustrative andexemplary and are not meant to limit the invention. Other embodimentscan be utilized and structural changes can be made without departingfrom the scope of the claims of the present invention.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “a particle” includes aplurality of such particles, and a reference to “a surface” is areference to one or more surfaces and equivalents thereof, and so forth.

The symbol “>” when used in the context of the wavelength ofelectromagnetic radiation, means “greater than or equal to”; the term“≧640 nm” means electromagnetic radiation having a wavelength of atleast or greater than 640 nm, for example, 640 or 641 nm.

The term “about” when used in the context of electromagnetic radiationwavelength means a wavelength of within 2 nm of the wavelength aswritten; therefore the term “a wavelength of about 640 nm” means theelectromagnetic wavelength is between 638 nm and 642 nm.

DETAILED DESCRIPTION OF THE INVENTION

The invention herein disclosed provides for devices and methods that maybe used for the synthesis of phycocyanins. The method results in agreater than 4.5-fold increase in phycocyanin levels, a clearimprovement over the prior art. The devices herein disclosed may be usedin many applications, including, but not limited to, use as a naturalfood colouring, as an antioxidant in the food supplement industries, inthe nutraceutical, pharmaceutical, and cosmeceutical industries, and asa non-toxic ink The invention provides improved methods for thesynthesis and commercial production of phycocyanins.

In an exemplary embodiment, the method includes providing amicroorganism capable of synthesizing phycocyanins, providing a suitableculture and growth medium, illuminating the microorganism in culturewith red and/or near-infrared light, and in the alternative,illuminating the microorganism in culture with red and/or near-infraredmonochromatic light. In an alternative embodiment, the method alsoprovides illuminating the microorganism in culture with white light.

In one embodiment the red light consists of electromagnetic radiationhaving wavelengths between about 640 nm and about 720 nm. In anotherembodiment the red light consists of electromagnetic radiation havingwavelengths between 640 nm and 1000 nm. In another embodiment the redlight consists of electromagnetic radiation having a maximum wavelengthemission of 680 nm. In an alternative embodiment the red light consistsof electromagnetic radiation having a wavelength of 678 nm In anotheralternative embodiment the red light consists of electromagneticradiation having a wavelength of 682 nm. In another alternativeembodiment the red light consists of electromagnetic radiation having awavelength of 690 nm. In another alternative embodiment the red lightconsists of electromagnetic radiation having a wavelength of 670 nm. Inan alternative embodiment the red light consists of electromagneticradiation having a mean wavelength of 680 nm, wherein the wavelength iswithin a 95% confidence interval of 640-720 nm. In one embodiment thewhite light consists of electromagnetic radiation having wavelengthsbetween 350 nm and 760 nm.

Culturing under red 680 nm LED light compared to white was shown toincrease PC production in A. platensis by an average of 5-fold and theseeffects could be seen visually in the cultures. Mass spectral analysishas shown some major differences and changes on the protein levelthrough culturing under the two different light sources. No significantdifference was seen in growth rate under the two light sources.

The invention will be more readily understood by reference to thefollowing examples, which are included merely for purposes ofillustration of certain aspects and embodiments of the present inventionand not as limitations.

EXAMPLES Example I Batch Cultures

F/2 sterile medium (CCAP [Culture Collection of Algae and Protozoa]recipe) supplemented with 2.5 g/l NaNO₃ (pH 8) was inoculated underaseptic conditions at 20% (v/v) with Arthrospira platensis (CCMP[Culture Collection of Marine Phytoplankton] 1295/Bigelow Laboratory US)(OD 0.11-0.12) in logarithmic growth phase. A stirred tankphotobioreactor (STPBR) (Infors Labfors 4 benchtop modified bioreactor)with either white (Lumitronix Barre LED High-Power SMD 600 mm, 12 V) or680 nm Red LEDs (FIGS. 2 and 3) was operated with 2.75 of culture at 30°C. and 45 μmol⁻¹ m⁻² light intensity with 18:6 light:dark cycle andimpeller speed 200 rpm with natural compressed air (˜0.03% CO₂) suppliedat 0.08 LPM (VVM (volume of air per volume of culture per minute) ˜0.03litres air per litres medium per minute, LPM) through a gauzed ringsparger. pH and dissolved oxygen was recorded online in 10 minuteperiods (Mettler Toledo probes). 8 ml samples were taken aseptically ondays 1 (inoculation), 3, 6, 7, 10, 13, and 14 for analysis.

Example II Growth Measurement

Optical density (OD) was used alongside chlorophyll autofluorescence(CF) and direct cell counts as a proxy for growth. OD was measured intriplicate at 750 nm Griffiths et al. (2011) [14] using a Cary 100UV/Vis Spectrophotometer (Varian) corrected with F/2 medium. CF wasanalysed in three triplicate 300 μl samples divided into individualwells of a black 96 well plate. Samples were excited at 430 nm andemission measured at 690 nm using a FLUOstar OPTIMA fluorescence platereader (BMG LABTECH). Readings were taken against blank samples of F/2medium and the average values in arbitrary fluorescence units used forstatistical analysis. Cell counts were performed using a Sedgewickrafter counting cell and using Leitz Dialux 20 light microscope.Triplicate 10 random sample counts were taken for 1 μl of culture.

Example III Morphological Assessment

The total length and width of the spirals of 20 cyanobacteria weremeasured to assess any changes in the morphological features of thetrichomes. Images were taken using Leitz Dialux 20 light microscope andEasyGrab software with analysis performed using Image J. Image size wascalibrated using graticules at 630 pixels mm⁻¹.

Example IV Phycocyanin Analysis

PC extraction was based on the method by Zhang and Chen (1999) [15]. 5mL samples were harvested by centrifugation at 3000 g/10 minutes (Sigma3K18C centrifuge) in pre-weighed glass tubes. Cells were washed once indeionized water and the wet biomass weighed. The pellet was thenresuspended in 3 mL 0.05 M sodium phosphate buffer (pH 7). Cells weredisrupted by a freeze/thaw cycle (−20 ° C.) over 1 hour and sonicatedfor 3 minutes at 6 microns amplitude (Soniprep 150, MSE). Samples werethen centrifuged at 10,000 g, 30 minutes (Sigma 1-15 microcentrifuge)and the absorbance of the supernatant scanned over 200-800 nm byspectrophotometer (Cary 100 UV-Vis spectrophotometer, Varian) using aquartz cuvette. Sodium phosphate buffer (0.05 M) was used as a blank andthe PC concentration and purity calculated using the method by Bennetand Bogorad (1973) [10] (Equation 1) and Boussiba and Richmond (1979)[16] (Equation 2) respectively. Extraction yield was calculated as belowin Equation 3. PC concentration was analysed at day 14 (or when growthreached OD 0.33) as three replicates.

PC(mg/mL)=(A620−0.474(A655))/5.34.  1.

PC purity=A620/A280.  2.

Extraction yield (mg PC/mg biomass)=(PC concentration*volume of solvent(mL)/biomass (mg)  3.

Example V Mass Spectrometry (MS) Analysis

Matrix Preparation

20 mg alpha-Cyano-4-hydroxycinnamic acid (HCCA) (Brucker Daltonics) wasmixed with 1 ml 50% acentonitrile: 2.5% TFA solution and saturated by 30minutes incubation at 25° C. in an ultrasonic water bath (Grantinstruments, Cambridge), vortexed at 15 minutes. Matrix was centrifuged(14,000 g, 1 minutes) (Sigma 1-15K microcentrifuge) and 50 μl aliquotsprepared fresh for use.

Sample Preparation:

1 ml samples were centrifuged (14,000 g, 5 minutes) (Sigma 1-15Kmicrocentrifuge) and the pellet washed twice in fresh deionized water(fdw) and stored frozen at −80° C. Pellets were thawed on ice andresuspended in 50 μl fdw before spotting. Samples were mixed 1:1 withHCCA matrix and 4 μl duplicate samples spotted onto a steel target plate(MTP 384 target plate ground steel, Brucker) along with 1 μl bacterialstandard (Brucker) layered with 1 μl HCCA matrix as a calibrant. Samplesthen underwent MS analysis (Bruker ultraflex II maldi-toftof). Spectrawere analysed using flexAnalysis software package (Bruker).

Example VI Population Analysis

Samples were frozen in 15% sterile Glycerol and frozen at −80° C. forpopulation analysis (Dr Andrew Free and Rocky Kindt, EdinburghUniversity).

Example VII Statistical Analysis

Data analysis was performed using Microsoft Excel 2007 and GraphpadPrism 5.

Example VIII Results: Growth

No significant difference in growth of cultures was observed under red680 nm compared to white LED light (FIG. 4). Note the largeacclimatization lag period in batch Red 2 (FIG. 4). The culture requireda period to acclimatize to be able to utilize the red 680 nm light inphotosynthesis (from observation), and this acclimatization wasreversible.

Example IX Results: PC Analysis

Phycocyanin absorbs at 620 nm. The presence of PC in the extracts of red680 nm LED batches compared to white LED was much higher (FIGS. 5 & 6).An interesting blue-shift was observed in the second Chlorophyll a peakaround 670-680 nm where the peak red 680 nm extract absorption is677-678 nm and the peak white extract absorption is 673-674 (FIG. 6).

PC concentration was increased 5-fold on average (at least nine samples)through culturing under red 680 nm compared to white LED light and therewas a slightly higher PC purity under red 680 nm LED light compared towhite (FIG. 7 table). Visual colour differences were observed in theculture most likely resulting from increased PC content of the cellscultured under red 680 nm light (FIG. 7).

Example X Results: MS Analysis

Whole cell MALDI spectra showed differences in abundant proteins fromculturing under red 680 nm compared to white LED light (FIG. 8).

Example XI Results: Leaching Differences

When discarding the samples prepared for MS analysis, a highconcentration of PC had leached into solution in the red 680 nm samples(FIG. 9). By eye the colour difference in leached PC was substantiallyhigher in the red 680 nm culture compared to white LED light, lookingmuch greater than a 5-fold increase. This indicated a possibledifference in the PC leaching characteristics in the red 680 nm culture,which may be beneficial to downstream processing (DSP). Culturing under680 nm light may increase the leaching of PC from the biomass.

Example XII Results: Culture Aggregation

Culturing under 680 nm light may also increase aggregation of theculture, with benefits to DSP. Aggregation may be a result of increasedproduction of extracellular polysaccharide (EPS) as a stress response.This is clearly an unexpectedly superior result that could not have beenpredicted by one skilled in the art.

Those skilled in the art will appreciate that various adaptations andmodifications of the just-described embodiments can be configuredwithout departing from the scope and spirit of the invention. Othersuitable techniques and methods known in the art can be applied innumerous specific modalities by one skilled in the art and in light ofthe description of the present invention described herein. Therefore, itis to be understood that the invention can be practiced other than asspecifically described herein. The above description is intended to beillustrative, and not restrictive. Many other embodiments will beapparent to those of skill in the art upon reviewing the abovedescription. The scope of the invention should, therefore, be determinedwith reference to the appended claims, along with the full scope ofequivalents to which such claims are entitled.

REFERENCES

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[2] Gonzalez, R. et al. 1999. Anti-inflammatory activity of phycocyaninextract in acetic acid-induced colitis in rats. Pharmacological Research39(1): 55-59.

[3] Romay, C. et al. 1998. Antioxidant and anti-inflammatory propertiesof C-phycocyanin from blue-green algae. Inflammation Research 47: 36-41.

[4] Pinero Estrada, J. E., Bermejo Bescos, P., and Villar del Fresno, A.M. 2001. Antioxidant activity of different fractions of Spirulinaplatensis protean extract. Il Farmaco 56: 497-500.

[5] Belay, A. 2002. The Potential Application of Spirulina (Arthrospira)as a Nutritional and Therapeutic Supplement in Health Management. TheJournal of the American Nutraceutical Association 5(2): 27-48.

[6] Kirk, J. O. T. 2000. Light & Photosynthesis in Aquatic Systems.Second ed. Cambridge University Press, Cambridge.

[7] Wang, R. T., Stevens, C. L. R., and Myers, J. 1977. Action spectrafor photoreactions i and ii of photosynthesis in the blue-green algaanacystis nidulans. Photochemistry and Photobiology 25(1): 103-108.

[8] Johnson, J.D. 2006. The Manganese-calcium oxide cluster ofPhotosystem II and its assimilation by the Cyanobacteria. 2006 (Lastaccessed 20 Jul. 2012). Available at:http://www.chm.bris.ac.uk/motm/oec/motmc.htm.

[9] Habib, M. A. B., Parvin, M., Huntington, T. C., and Hasan, M. R.2008. A review on culture, production and use of spirulina as food forhumans and feeds for domestic animals and fish. FAO Fisheries andAquaculture: Rome.

[10] Bennett, A., Bogorad, L. 1973. Complementary chromatic adaptationin a filamentous blue-green alga. The Journal of Cell Biology 58(2):419-435.

[11] Bogorad, L. 1975. Phycobiliproteins and complementary chromaticadaptation. Annual Review of Plant Physiology 26: 369-401.

[12] Walter, A. et al. 2011. Study of phycocyanin production fromSpirulina platensis under different light spectra. Brazilian Archives ofBiology and Technology 54: 675-682.

[13] Wang, C.-Y., Fu, C.-C., and Liu, Y.-C. 2007. Effects of usinglight-emitting diodes on the cultivation of Spirulina platensis.Biochemical Engineering Journal 37(1): 21-25.

[14] Griffiths, M. J., Garcin, C., van Hille, R. P., and Harrison, S. T.L. 2011. Interference by pigment in the estimation of microalgal biomassconcentration by optical density. Journal of Microbiological Methods85(2): 119-123.

[15] Zhang, Y.-M. and Chen, F. 1999. A simple method for efficientseparation and purification of c-phycocyanin and allophycocyanin fromSpirulina platensis. Biotechnology Techniques 13(9): 601-603.

[16] Boussiba, S. and Richmond, A. E. 1979. Isolation andcharacterization of phycocyanins from the blue-green alga Spirulinaplatensis. Archives of Microbiology 120(2): 155-159.

[17] Sudhir et al. 2005. The Effects of Salt Stress on PhotosyntheticElectron Transport and Thylakoid Membrane Proteins in the CyanobacteiumSpirulina platensis. Journal of Biochemistry and Molecular Biology 38:481-485.

[18] Verma, K., Mohanty, P. 2000. Changes of the photosyntheticapparatus in Spirulina cyanobacterium by sodium stress. Z Naturforsch C55: 16-22.

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1. A method for the synthesis of a phycocyanin, the method comprisingthe steps of (i) culturing a microorganism in a suitable growth mediumand (ii) providing a suitable source of light, wherein the lightconsists of electromagnetic radiation having a wavelength of between 640and 720 nm and wherein the microorganism in culture synthesizes aphycocyanin.
 2. The method of claim 1, wherein the microorganism isArthrospira platensis or Arthrospira maxima (Spirulina or Arthrospiragenus).
 3. The method of claim 1, wherein the electromagnetic radiationconsists of a wavelength of 680 nm.
 4. The method of claim 3, whereinelectromagnetic radiation consists of a wavelength of 682 nm.
 5. Themethod of claim 3, wherein electromagnetic radiation consists of awavelength of 678 nm.
 6. (canceled)
 7. The method of claim 1, whereinlevels of the synthesized phycocyanin are at least four-fold greaterthan levels of synthesized phycocyanin in the same microorganismseparately cultured in the presence of white light.
 8. The method ofclaim 7, wherein the white light consists of electromagnetic radiationhaving a wavelength of between 350 nm and 760 nm.
 9. The method of claim7, wherein the synthesized phycocyanin leaches from the microorganism.10. The method of claim 1, wherein the microorganism is substituted withanother organism, and wherein the other organism is selected from thegroup consisting of a photosynthetic bacterium, a photosyntheticarchaean, a photosynthetic protist, a photosynthetic alga, aphotosynthetic moss, and a photosynthetic plant.
 11. The method of claim10, wherein the organism comprises recombinant DNA.
 12. The method ofclaim 1, wherein the method is suitable for the synthesis of abiochemical, wherein the biochemical is selected from the groupconsisting of hyaluronans, glucosamines, saccharides, polysaccharides,phycobiliproteins, allophycocyanin, phycoerythrin, bilin, phycobilin,proteoglycans, and glycosaminoglycans.
 13. A system for producingphycocyanin, the system comprising a vessel and a lamp, wherein the lampgenerates electromagnetic energy consisting of a wavelength of 640 nm orgreater, and wherein the vessel further comprises a microorganismcapable of synthesizing phycocyanin.
 14. A system for producingphycocyanin, the system comprising a vessel and a lamp, wherein the lampgenerates electromagnetic energy consisting of a wavelength of 680 nm,and wherein the vessel further comprises a microorganism capable ofsynthesizing phycocyanin.
 15. The method of claim 14, wherein themicroorganism is Arthrospira platensis or Arthrospira maxima (Spirulinaor Arthrospira genus).